ILAR J
Volume 49, Number 2, 2008
Microbial Quality Control for Nonhuman Primates

Kemnitz and Mansfield.  Introduction: Challenges in Microbial Quality Control for Nonhuman Primates, pp. 133-136

Domain 1: Management of spontaneous and experimentally induced diseases and conditions

T1: Prevent spontaneous or unintended disease or conditions

Species: Nonhuman primates (general)

SUMMARY: While other species have had more success in microbial control/elimination of pathogens, this process has been more difficult in nonhuman primate colonies. The reason for this is likely due to multiple factors including: social interaction needs for normal primate behavioral development, housing and breeding strategies currently in use to promote this normal development, long life span of primates, ethical concerns of euthanasia of positive animals, and the fact that humans serve as carriers/vectors for reintroduction of pathogens.

Controlling the microbial status of primates is warranted when research can be affected in negative ways. These negative effects include zoonotic disease in humans, overt morbidity and mortality in research animals, or altered responses to experimental manipulation.

QUESTIONS:
1.	True/False: An exogenous pathogen generally cause little to no disease in an infected animal.
2.	Which of the following should be included in a microbial quality control program?
a.	Fecal culture
b.	Thorough necropsy at time of death
c.	Serologic evaluation for specific pathogens
d.	All of the above.
3.	Why is cesarean rederivation with separation of primates not a viable strategy for creating “clean” primate colonies?

ANSWERS 
1.	False. This describes an indigenous pathogen.
2.	D. also include: vigorous diagnostics to determine causes of illness, and integrated medical records, lab tests and housing records.
3.	Parental input is vital for normal primate social development.

Morton et al.  Specific Pathogen-Free Macaques: Definition, History, and Current Production, pp. 137-144

Domain 1: Management of spontaneous and experimentally induced diseases and conditions

Task 1: Prevent Spontaneous or unintended disease or condition

Primary species – macaque

SUMMARY: Current research using macaques, especially AIDS models, requires more stringent SPF conditions. Mycobacterium tuberculosis was the initial pathogen of concern. Four additional pathogens form the basis of most recent SPF colonies: simian type D retrovirus (SRV), simian immunodeficiency virus (SIV), simian T cell lymphotropic/leukemia virus (STLV), and Cercopithecine herpesvirus 1 (CHV-1). This article reviews the disease presentation, transmission, testing and preventive strategies of these most commonly targeted pathogens.

All research colonies should be tuberculosis-free and any SPF colony is defined as SIV-free. Further levels add status as follows:

1	Level 1: SRV-free
2	Level 2: SRV-, STLV-free
3	Level 3: SRV-, STLV- and CHV1-free
4	Level 4: all above pathogens and additional virus exclusions (i.e., simian foamy virus or other herpesviruses)

Mycobacterium tuberculosis
1	Recently emerged as a common pathogen in imported Asian macaques.
2	The disease is highly contagious and sometimes rapidly fatal, and latent infections are common.
3	Mammalian tuberculin skin testing of all colony animals is the status quo.
4	New assays include whole blood in vitro gamma assay and rapid lateral flow test of M. tuberculosis-specific proteins.
5	The most effective testing regimen includes routine skin testing and one of these new assays in addition to tradition diagnostics such as thoracic radiography, BAL.

Retroviruses
1	SIV
o	Not naturally present in wild populations of Asian or Indian macaques but can readily infect them with experimental exposure.
o	Most infected animals rapidly progress to terminal disease.
o	Closely related to HIV and presents a potential human health risk.
o	Rare nonexperimental horizontal transmission has occurred in research colonies, so infected animals should be culled immediately and SIV monitoring continued regularly in designated SPF colonies.
o	Screening tests should react with all SIV variants. Immunoassays should use whole virus preparations that target conserved regions of the genome such as the p27 gene.
2	SRV
o	Exists in most macaque research colonies in the US and Europe.**
o	Does not cause AIDs-like disease similar to SIV but can confound research results in immunocompromised NHPs.**
o	Productive acute infections with high levels of virus in body fluids result in effective horizontal and vertical transmission.
o	Most animals mount an effective antibody response against the outer membrane glycoprotein pg70 within 6 weeks of exposure. In some animals this virus suppression is only temporary which can lead to subsequent shedding during times of stress or immune suppression.
o	Five serotypes have been identified with cross reactivity at major capsid protein p27 and transmembrane protein gp20-22. Different serotypes are more common in different species of macaque (1, 3 and 5 in M. mulatta and 2 in M fascicularis). Serologic tests should target these proteins.
o	Small numbers of animals are viremic without high circulating antibodies, so screening must include serology and direct virus testing.
o	Endogenous retroviruses are present in the cells of macaques that are related to SRV, so PCR primers must be validated with DNA sequencing when tests are positive to confirm the presence of exogenous SRV rather than endogenous viruses.
3	STLV
o	Usually subclinical, infection is latent.
o	Closely related to human T lymphotrophic/leukemia virus (HTLV) and presents a potential human health risk.
o	Genomes of both HTLV and STLV contain regulatory proteins that have been shown in HTLV to interfere with T-cell regulation.
o	Low natural prevalence (5-40%).
o	Most mature virus is contained within cells which results in a low transmission frequency in group-housed animals and also no transmission among individually housed animals.
o	Diagnostic tests include antibody tests against HTLV and PCR that use HTLV sequences.

Cercopithecine Herpesvirus 1
·	Primary occupational health concern for personnel working with macaques.
·	Enters oral or vaginal mucosa resulting in a lifelong infection in the trigeminal and lumbosacral sensory ganglia. Most new macaque infections occur at the onset of sexual maturity.
·	Latent periods of infection with no clinical signs.
·	Screening tests include herpes simplex virus (HSV)-based ELISA and antibody tests against baboon alpha herperviruses, but no CHV-1-specific test is available. Test sensitivity should exceed test specificity to reduce the chance of false negatives.
·	CDC select agent, level 4 risk designation for propagation

SPF Colony Management Recommendations
1	SPF 1 and 2
o	All new animals are quarantined pending 2 consecutive negative tests 6 weeks apart for SRV, STLV and SIV. Grouping should remain constant during this time.
2	Antibody test preparations should contain glycoproteins and major capsid proteins of the viral serotypes most prevalent in the animals to be screened.
2	A whole blood method should be used to detect viral DNA and RNA for all serotypes present in the animals to be screened.
3	Infectious SRV can be cultured in Raji cells to detect latent infections.
o	Resident animals should be tested semi-annually and 6-8 weeks after contact or potential contact with any new animals.
o	Dams and infants should be tested 6-8 weeks after birth, juveniles should be screened before being moved and 4-6 weeks after rehousing.
2	SPF 3
o	SPF 2 status
o	Juveniles that are seronegative for CHV-1 are segregated from adults and retested at sexual maturity. These animals are used as colony founders as they are presumed to produce seronegative offspring. Any new animals must be derived in the same way.
o	Frequent testing and immediate removal of seroconverts.
o	Strict husbandry procedures and stable groupings to avoid inadvertent infection.

QUESTIONS:
1. 	Which virus is excluded from all SPF levels for macaques?
a. 	SRV
b. 	Simian foamy virus (SFV)
c. 	CHV-1
d. 	SIV
2. 	Whole blood in vitro gamma assay and rapid lateral flow test for ESAT-6 and MPB83 are newer assays to detect which pathogen in macaque colonies?
a. 	SIV
b. 	CHV-1
c. 	Mycobacterium tuberculosis
d. 	None of the above
3. 	Which virus excluded in SPF macaque colonies is closely related to endogenous retroviruses, complicating PCR analysis?
a. 	SIV
b. 	SRV
c. 	CHV-1
d. 	STLV
4. 	What is the scientific name for herpes B virus?
5. 	When do most new macaque infections of herpes B virus occur?
a. 	At birth through vertical transmission
b. 	During times of increased stress
c. 	At the onset of sexual maturity
d. 	None of the above
6. 	A macaque colony that is SPF 2 excludes which viruses?
a. 	SIV and CHV-1
b. 	SIV and SRV
c. 	SIV, SRV and CHV-1
d. 	SIV, SRV and STLV
7. 	Which SRV serotype is most common in Macaca fascicularis?
a. 	SRV-4
b. 	SRV-2
c. 	SRV-3
d. 	SRV-1
8. 	Antibodies against major capsid protein p27 and transmembrane protein pg20-22 are used in testing for which SPF 2-excluded pathogen?
a.	SRV
b. 	SIV
c. 	STLV
d. 	M. tuberculosis
9. 	STLV infection is characterized by the following:
a. 	An AIDs-like syndrome
b. 	Latent infection of the trigeminal ganglion
c. 	A low transmission rate among group-housed animals
d. 	None of the above

ANSWERS: 
1. 	d 
2. 	c 
3. 	b 
4. 	Cercopithecine herpesvirus 1
5. 	c
6. 	d
7. 	b
8. 	a
9. 	c

Roberts and Andrews.  Nonhuman Primate Quarantine: Its Evolution and Practice, pp. 145-156

Domain 1:  Management of Spontaneous and Experimentally Induced Diseases and Conditions.  Task T1-T3: Prevent, control, and diagnose spontaneous or unintended disease or condition.

Domain 4:  Animal Care. Knowledge:  Containment facilities; animal procurement considerations; methods of sterilization, sanitation, and decontamination.

Domain 5:  Regulatory Responsibilities ACLAM Knowledge: laws, regulations, policies, and standards; BMBL; Endangered Species Act/CITES; IATA;

Quarantine History: Quarantining of NHPs began in the 1940s and 1950s as increasing numbers of NHPs were being imported for disease research.  Many of these animals were wild caught and the potential for the introduction of novel agents that could pose a threat to human and animal health existed.

US NONHUMAN PRIMATE IMPORTATION & QUARANTINE PRACTICES

Importation and Infectious Disease: Basic principles of NHP quarantine were identified in the 1950s as the national effort to develop a polio vaccine required the importation of more than 200,00 rhesus monkeys annually for 6 years.

The stress of capture, transport, and exposure to human pathogens resulted in epizootics of infectious disease in newly imported populations.  The disease outbreaks led to loss of animals, poor animal quality, and compromised research programs.

To address these problems government programs and research facilities began developing conditioning programs in source countries and quarantine programs in receiving facilities to help detect the presence of infectious disease and its spread in captive primate populations.

"All in and all out" philosophy expounded.  Karl Habel in 1947 stated (referring to rhesus importation) large groups should be kept "as homogeneous as possible", "All animals in a room should be used before more are moved in" and that "new monkeys should be isolated" and only allowed to into another colony after having passed "two tuberculin tests one month apart."

Despite this advice, disease outbreaks occurred in domestic colonies due to mixing of primate species in transit or inadequate separation at the receiving facility.  Examples are given of the dramatic effects when mixing populations of different species from the same geographic region (e.g., Squirrel monkeys with Herpes tamarinus) and mixing different species from different continents (e.g., many African primate species carry simian hemorrhagic fever which causes a severe hemorrhagic crisis, leading to organ failure and death in aberrant hosts).

Institutional Quarantine: NHP in the US are subject to both primary (aka international, refers to when they enter the US from a foreign country) and secondary (aka domestic, isolation upon transfer between institutions within the US) quarantine.

Until recently the length of quarantine was determined by the institution and varied widely.  At a minimum, the practice of requiring 3 negative TB skin tests 2 weeks apart necessitated a period of at least 31 days.

Government Regulation (1975-1980): Marburg Outbreak - Occurred in 1967 in Marburg, Germany.  Importation of African green monkeys resulted in transmission of a previously unrecognized filovirus to laboratory workers that participated in necropsy and tissue harvest.  7 of 27 primary human cases died and 6 other people were infected by secondary contact.  The Marburg animals had been used shortly after arrival with no quarantine period.

The Marburg outbreak and concerns about the risks of other potential zoonotic agents prompted the US Public Health Service (USPHS) to call for national regulations for animal quarantine.  National regulations were determined to be preferable to individual state regulations which might result in varied practices and confusion.

Regulations enacted in October 1975 established basic quarantine requirements and required notification to the CDC of any cases of Marburg, yellow fever, or monkeypox in any imported NHPs.  Can't import NHPs for pets.  Required facilities that import NHP to 1) register with the CDC, 2) certify that imported NHPs are used for scientific, education, or exhibition purposes, 3) Isolate shipments for 31 days (3 TB skin tests at 3-wk interval, and incubation period of known serious infectious zoonoses), 4) maintain records for each shipment, 5) report suspected zoonotic illness in animals or workers.

National Research Council's (NRC) Committee on NHP in 1980 established the standard method for screening NHPs for TB; noncompliant facilities were not allowed to import NHPs. "A suspension (0.1mL) containing >= 1500 tuberculin units of USDA veterinary tuberculin should be injected intradermally in the edge of the upper eyelid with a sterile 25-27 gauge needle," followed by a daily visual examination for 3 days with a skin reaction rating of 1 to 5.  Any animal that tested positive was euthanized and the remaining animals in that shipment had to complete 5 additional negative TB skin tests prior to release.  

Lessons Learned in Primate Importation and Infection Control (post-1980):  A cluster of 4 human cases of Herpes B virus (Cercopithecine herpesvirus 1) in 1987, including 2 deaths, reinforced the necessity of certain safety precautions and the importance of appropriate infection control practices (beyond quarantine) for NHPs.  CDC came out with a set of guidelines in 1987 to prevent the transmission of B virus to humans and to improve general infection control in NHP facilities.

Two outbreaks of hemorrhagic disease occurred in quarantined cynomolgus macaques occurred in 1989.  The first outbreak was determined to be caused by simian hemorrhagic fever (SHF) which is not zoonotic, but has a clinical presentation in animals similar to Ebola and Marburg viruses.  Its presence in the animal facility raised concerns about the possible presence of a more serious human outbreak and the spread across groups of primates in separate quarantine areas  suggested lapses in facility design and/or infection control practices.  The second involved the "Reston agent", an Ebola-like agent, and prompted a CDC review of quarantine practices.

CDC's Response to the Outbreaks (1990-1993):  Upon surveying other NHP quarantine facilities, the CDC found the Ebola-like agent in another NHP facility. CDC initiated a comprehensive review of quarantine facility practices and standard operations.

Issued Interim Guidelines for Handling NHP during Transit and Quarantine in January 1990. The article reproduces the 3 sets of guidelines.

Established a Special Permit for Importation program with additional control measures for the importation of rhesus, cynomolgus, and African green monkeys. 

CDC issued Interim Guidelines for Tuberculin Skin Testing of NHPs during Quarantine in 1993 in response to TB positive cynomolgus monkeys imported from Mauritius and a subsequent review of NHP importations for a 3 year period starting in June 1990.

Impacts of CDC 1990 Guidelines: A reduction from 140 NHP CDC-registered import facilities in 1989 to 27 in 1999.  A reduction in NHP mortality rates during shipment and quarantine from ~20% before the Special Permit to < 1% by 1999. 

NHP Sources and Quarantine Facilities: Many primate source countries now only export the conditioned offspring of animals maintained in captivity.  Conditioning programs coupled with screening for pathogens, decrease the rate of morbidity and mortality in transported animals.

ELEMENTS OF A NHP QUARANTINE PROGRAM

Quarantine Facility: Facility that allows for physical segregation of the quarantined population including air space, wastes, body fluids, and tissues.  Ideal is complete physical separation in a dedicated building; rarely an option b/c of space and expense limitations.

If the quarantine area is located in a multiuse building the following measures are essential to contain or minimize risk of infection - 1) limit access to appropriate personnel; 2) anterooms; 3) doors with windows for observation prior to entrance; 4) rooms meets standard design requirements for NHP housing.

The containment facility design must meet ABSL-2+ indicating a need for ABSL-3 PPE and procedures.

Health Screening and Animal Husbandry: A primary quarantine program should include at a minimum: 1) daily observation of the animal, its wastes and food consumption; 2) 3 consecutive negative TB skin tests at 2-wk intervals; 3) isolation for 31 days; 4) physical exam with additional weighing during immobilizations for TB tests; 5) baseline blood values; 6) parasitological examination; 7) rectal culture.

2 to 3 day acclimation period prior to immobilization for physical exam and TB tests.

Singly housed in squeeze back cages to facilitate safer observation and chemical observation.

Animal Illness and Necropsy: Animals typically not removed from quarantine area.  This restriction may limit treatment and diagnostic options.  It is ideal to have dedicated clinical equipment in biocontainment area for analysis of samples.

Personnel Training and Occupational Health Standard PPE includes 1) moisture resistant protective clothing; 2) double protective gloves; 3) NIOSH-approved N95-rated disposable particulate respirator; 4) protective footwear; 5) Face shield to prevent splash contact with eyes and mucous membranes.

Staff training on donning and removing PPE as well as other correct work practices.  
All personnel should be trained on proper use and disposal of sharps.

All individuals working in NHP quarantine should enroll in the facility's occupational health program.  All new employees should complete an initial health evaluation including a TB skin test (or chest radiograph) and appropriate immunizations.  Follow-up health assessments and TB tests should be repeated on a regular basis. Monitor and record employee illness and injury.  

Quarantine Disease Surveillance: Address both agents that cause clinical disease and those that represent possible confounders to research.

Tuberculosis: TB skin test has significant limitation.  False positives can result from poor technique and other issues of sensitivity and specificity. The most significant challenge to primate importation facilities is the screening process for TB.  Development of a rapid, cost-effective in vitro diagnostic assay of infection with mycobacterial agents is needed to improve the efficiency of NHP quarantine.  

Enteric Pathogens: Outline procedures to screen for bacterial, protozoan, and metazoan pathogens.  Usually possible to treat infections in quarantine.  Some facilities routine treat animal prophylactically for parasites.

Viral Infections: Challenge is to improve diagnostic tools available for effective and reliable identification of animal infected with undesirable viral pathogens.

The liver from any Special Permit species with suspect illness or death is tested for the presence of filovirus.  If any animals in a shipment exhibit signs suggestive of a hemorrhagic disorder, serum must be collected from all animals in that shipment for IgG ELISA Ab test for filovirus.

Expansion of viral screening in domestic breeding colonies to include viral pathogens that may be asymptomatic but may represent research confounds (i.e., SRV, STLV-1, SFV, SIV in macaques).  

Institutional and national programs working to create SPF primate colonies have arisen. 

QUESTIONS:
1.  	What species are included in the Special Permit for Importation program?  What is the rationale for including these species?
2.	What was the principal incident that prompted a CDC review of quarantine procedures and ultimately led to the issuance of "Interim Guidelines for Handling NHPs during Transit and Quarantine" and the Special Permit for Importation program in 1990?
3.  	List the guidelines and regulations that provide specifications for animal crates used to ship NHPs.
4.  	What is the minimum quarantine period and what it based on?
5.	After the removal of the last animal with a positive TB test, how many additional negative TB skin tests do animals from that cohort have to have before being released from quarantine?

ANSWERS:
1.	Rhesus macaques, cynomolgus macaques, and African green monkeys.  The measures focus on these species because the demonstrated seroreactivity to filoviruses and represented the vast majority of NHPs entering the country at the time.
2.  	Outbreak of an Ebola-like filovirus in cynomolgus macaques in Reston, VA in 1989 and subsequently finding the agent in another facility.  
3.  	Special Permit program, Animal Welfare Act, International Air Transport Association
4.  31 days.  This is the time required to have three negative TB skin tests spaced three weeks apart and covers the incubation period of known serious infectious zoonotic diseases.
5.  	5 additional TB skin tests

Simmons.  Development, Application, and Quality Control of Serologic Assays Used for Diagnostic Monitoring of Laboratory Nonhuman Primates, pp. 157-169

Domain 1: Management of Spontaneous and Experimentally Induced Diseases and Conditions; TT1.11. preventive medicine (e.g., immunization; quarantine; prescreening tests); TT1.12. diagnostic procedures b. serologic, cytologic, and molecular diagnostic tests (e.g., PCR; ELISA; IFA; HAI; MAP) and proper sampling techniques 

Primary species – nonhuman primates 
Secondary species – nonhuman primates 
Tertiary species – nonhuman primates 

SUMMARY: Serological assays able to provide reliable results are essential in management of nonhuman primates. The paper describes how serological assays are developed, including calculation of number of positive serum samples needed to validate it. Sensitivity and specificity are described and formulas to calculate these values are presented. Determination of assay cutoffs is done using nonparametric statistics, receiving operating characteristic (ROC) curve, or empirically. Quality controls in order to track assay performance need to be in placed and are described. Also, QA allows an assay to provide results that are accurate and precise. Serologic assays need to be monitored for variations over time, and this monitoring includes checks for random and systemic errors.

For interpretation of results from a serologic assay or diagnostic results, it is important to know the prevalence of the disease, sensitivity and specificity of the assay, and positive and negative predictive values (PPV and NPV) of the assay. Lastly, all unexpected results should be confirmed by additional diagnostics before a decision is made for the animal in question.

QUESTIONS: 
1. 	T/F. ELISA or the Luminex xMAP®-based multiplexed fluorometric immunoassay (MFIA) are commonly used as screening tests. 
2. 	T/F. Indirect fluorescent antibody (IFA), western immunoblot (WIB), or polymerase chain reaction (PCR) are commonly used as confirmatory assays. 
3. 	T/F. Antigens may include purified whole virus lysates, 1 or more immunodominant, or recombinant proteins. 
4. 	T/F. The least desirable positive serum samples are those from vaccinated animals, as they do not represent a normal process of seroconversion and may result in the production of interfering antibodies. 
5. 	When positive serum from vaccinated animals are appropriate. 
6. 	T/F. Analytical sensitivity is the lowest amount of antibody that can be detected by the assay, also called the assay’s limit of detection (LOD). 
7. 	T/F. Analytical specificity is an assessment of the selectivity of the antigen-antibody response and is often determined by performing the assay using a panel of heterologous, monotypic, positive control sera 
8.	When an assay is considered analytically specific? 
9. 	T/F. Cutoffs for a serological assay can be determined by nonparametric statistics, receiver operating characteristics (ROC), and empirically. 
10.	T/F. Assay validation is a formal process for determining the suitability of a given laboratory method for generating the data necessary to calculate assay performance characteristics and to develop an assay validation report. 
11.	T/F. Data from the validation study that agree with the known positive and negative status of the sera in question are classified as true positive (TP) and true negative (TN). 
12.	T/F. Sensitivity can be calculated using the formula: TP / (TP+FN), where TP= true positive and FN= false negative. 
13.	T/F. Specificity can be calculated using the formula: TN / (TN+FP, where TN= true negative and FP= false positive. 
14.	T/F. The goal of a quality assurance program is to enhance the confidence of both the laboratory and the consumer in the reported diagnostic test results. 
15.	T/F. Positive predictive value (PPV) can be calculated with the formula: TP / (TP+FP), where TP= true positive and FP= false positive. 
16.	T/F. Negative predictive value (NPV) can be calculated with the formula: TN / (FN+TN), where TN= true negative and FN= false negative. 
17.	T/F. Internal positive controls allow for more confidence in the validity of negative results when data are interpreted since they identify problems in an assay system that could produce false negative results. 
18.	T/F. The sample suitability controls indicate whether an animal has developed antibodies against cell culture proteins, insect cells, bovine serum albumin, or other contaminating cell culture components, any of which could result in a false positive assay response. 
19.	T/F. Precision1 is a measure of the reproducibility of a serodiagnostic test result and often is used as an indicator of the amount of random error in the system. 
20.	T/F. Precision is the same as accuracy. 
21.	T/F. Accuracy is the ability of an assay to identify sample results correctly. 
22.	T/F. Random error is the result of erratic runto-run variations in a diagnostic method that has a wide variety of sources and can be identified by a lack of precision (fluctuation) in assay controls over time. 
23.	T/F. Systematic error is a sign of a consistent positive or negative bias in the assay results. 
24.	T/F. Westgard’s rules include additional control limits at ±1 and ±2 SD that enable the laboratory to monitor control values for both dispersion and trends in the data that may develop over time. 
25.	T/F. when applying control rules to clinical data it is possible that random chance may result in the false rejection of data that are actually in control. 
26.	What is the likelihood of falsely rejecting a control run using a single control rule? 
27.	T/F. Interpretation of the quality control data will determine whether the assay run is in control and valid, or out of control and not valid. 
28.	T/F. The PPV is the probability that an animal that tests positive for an infectious agent or disease is truly infected with the agent. 
29.	T/F. The NPV is the probability that an animal that tests negative for an infectious agent is actually normal or not infected by the agent. 
30.	T/F. Serodiagnostic tests that often have greater diagnostic specificity (DSp), and thus a lower rate of false positives (FP) results, include IFA, WIB, hemagglutination inhibition, and PCR. 
31.	T/F. When the prevalence of an agent in a population falls to zero, the positive predictive value (PPV) drops to zero as well, and the negative predictive value (NPV) increases to 100%. 
32.	T/F. When the prevalence of an agent in the population approaches 100% so does the PPV for that population, while the NPV falls to zero. 

Answers:
1. 	True 
2. 	True
3. 	True 
4. 	True 
5. 	When the assay is intended to monitor seroconverted vaccinated animals 
6. 	True 
7. 	True 
8. 	When an assay is challenged with heterologous positive sera, and it does not react. 
9. 	True 
10. True 
11. True 
12. True 
13. True 
14. True 
15. True 
16. True 
17.	True 
18.	True 
19.	True 
20.	False 
21. True 
22. True 
23. True 
24. True 
25. True 
26. 0.27% 
27.	True 
28. True 
29. True 
32. True
31. True 
30. True 

Lerche et al.  New Approaches to Tuberculosis Surveillance in Nonhuman Primates, pp. 170-178

Domain 1: Management of Spontaneous and Experimentally Induced Diseases and Conditions 

Task 3: Diagnose disease or condition as appropriate 

Primary species: Nonhuman primate
 
SUMMARY: This article discusses advances made in the diagnosis and surveillance of TB in NHPs, and compares newer serologic tests to the traditional TB skin test (TST). In NHPs, TB is usually caused by Mycobacterium tuberculosis (M. bovis less commonly). The TST, which utilizes mammalian old tuberculin, is the standard for diagnosis and screening of TB in NHPs and is the only test approved by ILAR/CDC for use in primary import quarantine. The TST relies on a delayed-type hypersensitivity reaction (cell-mediated) and the main limitation is that the test will give only intermittent positive results in serial testing, and is not very accurate in diagnosing latent infections.

Newer assays of cellular immune responses rely on the production of interferon-gamma (IFN-gamma), and include PRIMAGAM and ELISPOT, although these assays share the same limitations as the TST. In the PRIMAGAM assay, whole blood is incubated with purified protein derivative of M. bovis (bPPD) or M. avium (aPPD) and other controls, and after 24 hours of incubation, IFN-gamma is  measured using optical density (OD). A + test is: (OD bPPD  OD aPPD) greater than 0.05 OD units. A stronger reaction to aPPD than to bPPD is often interpreted as sensitization to M. avium.

In vitro assays of humoral immune responses to TB antigens are also being produced. The main antigen targets used in these assays are ESAT-6 and CFP-10. Three assays have been produced: the multiplex microbead immunoassay (MMIA), the multiantigen print immunoassay (MAPIA) and the rapid test lateral flow immunoassay (e.g. Prima-TB STAT-PAK) and all rely on the incubation of serum with TB antigens followed by some type of detection/visualization.
 
QUESTIONS
1. 	Are all species of NHPs susceptible to TB? Which NHPs are the most and least susceptible?
2. 	How many reaction grades of the TST are there? Which grades are positive? Which grades are negative?
3. 	What is the length of the CDC-mandated primary import quarantine? How many consecutive negative TB tests are required during quarantine? How often are tests administered?
 
ANSWERS
1. 	Yes. OWM are most susceptible, NWMs are the least. Apes are intermediate.
2.	There are 5 grades. Grades 0-2 are -, grade 3 is suspect, and grades 4-5 are +
3. 	31 days. Need 3 consecutive negative TB tests at two week intervals. 

Sasseville and Diters.  Impact of Infections and Normal Flora in Nonhuman Primates on Drug Development, pp. 179-190

Domain 1:  Management of Spontaneous and Experimentally Induced Diseases and Conditions.
Task T1-T3: Prevent, control, diagnose spontaneous or unintended disease or condition.

Domain 4:  Animal Care. Knowledge:  Containment facilities; animal procurement considerations.

SUMMARY: Preclinical safety studies required for the approval of a pharmaceutical include single and repeat dose studies in two species to assess reproductive toxicity, genotoxicity, local tolerance, and carcinogenicity.  Cynomolgus and rhesus macaques are the two most common NHPs used by pharmaceutical companies.  

Infections: Most endemic infections in NHPs are clinically inapparent and self-limiting in immunocompetent animals.  They do not affect in-life or clinic-pathologic endpoint interpretations during routine drug safety studies.  Exceptions are infections associated with chronic enterocolitis, which is a widespread problem in macaques.  Immunosuppression, either naturally (i.e. retrovirus) or experimentally (i.e. irradiation, immunomodulatory agents, or chemotherapeutics) can make a previously unexposed animal more susceptible to primary outbreaks or recrudescence of these pathogens.

Viruses
1.	Retroviruses (Simian Type D retrovirus, Simian immunodeficiency virus, and Simian T Lymphotropic virus)

Simian Type D Retrovirus (SRV) historically had an impact on the drug industry because SRV-infected animals can become viremic yet remain antibody negative. Facilities had infected animals up until the 1990’s when virus isolation techniques along with serologic screening and PCR helped establish SRV-free colonies. The most common manifestation of SRV is weight loss, diarrhea, decreased lymphocyte and RBC counts, and an increase in the incidence of lymphoid infiltrates in various organ systems. SRV in its severe form can cause an immunodeficiency syndrome with opportunistic infections, inflammatory, and neoplastic/proliferative diseases.  Viral positive animals used in drug safety studies, SRV infection could be confused with a drug related effect.  SRV is now a rare occurrence and the validity of studies with viral positive animals should be questioned.
 
Simian Immunodeficiency Virus (SIV) naturally occurs in African NHPs, not Asian macaques, and is asymptomatic in the natural hosts.  In Asian macaques, SIV induces a lentiviral inflammatory and immunosuppressive AIDS-like disease.  SIV was detected in 1984 in captive rhesus macaques, and probably came from inadvertent infection from wild-caught sooty mangabeys.  Natural cross-species transmission is rare and has not impacted drug safety studies.  Efficient screening practices have created SIV-free colonies.

Simian T lymphotropic virus 1 (STLV-1) belongs to the primate T lymphotrophic group of retroviruses (STLV-2, STLV type L, STLV-3, and human counterparts). STLV is endemic in many wild caught and captive Asian and African monkeys and apes. STLV-1 is a cell-associated virus with tropism for CD4+ and CD8+ cells with transmission from cells of infected animals. Most infections are asymptomatic, but some animals may develop T cell lymphoma or lymphoproliferative disease.  Disease only occurs in African monkeys, but altered cytokine profiles have been reported in macaques, which can be a confounding variable. Screening practices have led to STLV-free colonies.

2.	Measles

Usually spread from infected humans. Very contagious and spreads rapidly, with symptoms ranging from fever, rash, and conjunctivitis to mortality secondary to transient immunosuppression.  PCR, virus culture, IgG and IgM levels can assess exposure to measles.  Animals that are exposed have life-long immunity, so quarantine and vaccination are effective for prevention.

3.	Opportunistic viral infections (OIs)

Common OIs I macaques include cytomegalovirus, adenovirus, simian virus 40, rhesus rhadinovirus, and lymphocryptovirus. These are most often associated with immunosuppressive retroviruses, irradiation, or drug-induced immunosuppression.  Less common infections include simian varicella virus, B virus, and simian parvovirus.  It is important to consider that the appearance of an infection may be atypical due to a diminished immune response, and may also depend on whether it is a primary infection or reactivation.     

4.	Hepatitis A Virus (HAV)

A small RNA picornovirus that is spread by the fecal-oral route and can infect a variety of old and new world NHPs including great apes, cynomolgus, rhesus and stump-tailed macaques, owl monkeys, marmosets, and baboons.  Natural transmission between animals has been reported in both the wild in the lab environment. Infection in macaques is generally mild and subclinical, with slight increases in ALT and AST, and mild changes in the liver.  HAV infection can alter cellular antiviral defense mechanisms. In both cynos and rhesus, infections are rarely persistent, but there may be periodic exacerbations with ALT elevation, viral shedding, and morphologic changes in the liver. Prophylactic HAV vaccination of NHPs used for toxicology studies is common. HAV in humans is distinct from simian HAV.  

Bacteria
1.	Camylobacter coli, Campylobacter jejuni, Shigella flexerni, Yersinia enterocolitica

Chronic gastritis and enterocolitis, either asymptomatic or with chronic diarrhea, are common in rhesus and cynomolgus macaques.  Studies indicate that one or more of a variety of bacterial species may cause chronic diarrhea. Campylobacter spp. have been associated with acute intestinal disease in NHP, and Shigella is associated with hemorrhagic diarrhea and death in NHPs.  Shigella can be controlled with antibiotics, but the presence of asymptomatic carriers makes colony eradication difficult.  Shedding can increase under stress or immunosupression.  Yersinia can cause outbreaks of diarrhea, but its incidence is usually lower than Shigella.  Each of these species has zoonotic potential. Sporadic outbreaks of chronic diarrhea of unknown etiology remain a problem in drug development, and can affect the immune system with increased cytokines and activated CD4+ and CD69+ T lymphocytes in the gut. Diarrhea in NHPs after a study has begun can adversely affect interpretation of results.

2.	Helicobacter pylori

Helicobacter pylori is generally asymptomatic but has been associated with gastritis and ulcers in humans.  Natural infection in macaques consists microscopically of epithelial hyperplasia, lymphoplasmocytic infiltrates and erosions of the stomach.  H. pylori should be differentiated from the larger H. heilmannii which is found in gastric glands, but has an unclear association with inflammation. 

3.	Tuberculosis

TB is caused by mycobacterium tuberculosis, and although infections are now greatly lowered due to surveillance, it remains a concern in NHPs.  TB infections in macaques used for drug studies are rare. TB skin tests at 2-3 week intervals over a 10 week quarantine period remains the gold standard.  

4.	Moraxella (Branhamella) catarrhalis

M. catarrhalis is part of the normal flora of the nasopharynx, but has been associated with outbreaks of epistaxis (“bloody nose syndrome”) in both healthy and immunocompromised macaques.  Outbreaks are usually mild and self-limiting, and tend to occur in winter when humidity is lower. Immunomodulatory drugs may increase incidence and severity of Moraxella outbreaks.

5.	Opportunistic bacterial infections

Infections associated with immunosuppressed animals include Mycobacterium avium complex (MAC), Rhodococcus equi, and enteropathogenic E. coli (EPEC). MAC is the most common disseminated bacterial infection in untreated SIV-infected macaques.  Because MAC organisms are ubiquitous in the environment, macaques are continuously exposed. MAC presents as focal to diffuse infiltrates of epithelioid macrophages with acid-fast bacilli in the lamina propria and submucosa of the GI tract and in sinuses of lymph nodes, and as pyogranulomas in the liver.  MAC lesions should be differentiated from those due to R. equi, which can disseminate from the lung to the intestinal tract in immunosupressed macaques and has a similar microscopic appearance to MAC.  EPEC has been observed in 20% of normal healthy adults, with diarrhea associated with SIV co-infection. Normal skin bacteria staphylococcus sp. and streptococcus sp. may invade skin wounds and cause abscesses and sepsis in immunocompromised NHPs.

Parasites
1.	Malaria

Malaria is a widespread disease cause by protozoans of the genus Plasmodium.  Monkeys can be the reservoir for human infection in enzootic areas, and in lab settings, malaria is present in NHPs imported from areas where the disease is endemic, such as South America, Africa, and Asia.  Natural infections in cynos and rhesus macaques may be caused by P. coatneyi, P. fragile, P. knowlesi, P. inui, and P. cynomolgi. The species of monkey and the Plasmodium spp. determine the individual disease outcome.  The source of primate can also influence the susceptibility to Plasmodium spp.  Infections with multiple Plasmodium spp. are common.  Splenectomy or immunosuppresion may enhance susceptibility and infection.  Plasmodium has a sexual development stage in the mosquito and an asexual stage in tissue cells and erythrocytes in the vertebrate host.  The greatest damage occurs during schizogany in the blood, which causes lysis of RBCs.  Both latent and active infections can cause immunologic effects and alterations of cytokines, thus infection free animals are advised for drug studies.

2.	Metazoan parasites

Most helminth parasite infections have been greatly reduced with the onset of antiparasitic treatments.  The exception is Strongyloides fulleborni, which persists despite prophylactic and therapeutic use of ivermectin.  Symptoms include cough, dyspnea, dermatitis, and diarrhea.  Trichuris trichiura has also been associated with significant clinical disease.  Schistosomiasis is a prevalent disease among humans and primates, with S. japonicum, S. mansoni, and S. hematobium of most importance.  The intermediate host needed to complete the life cycle is not present in the US.  Adult schistosomes cause little damage to the host, but the eggs that are released and fail to penetrate the vessel wall cause the granulomatous inflammation associated with the disease.  Rhesus macaques infected with S. mansoni showed significantly increased SHIV replication, and juvenile rhesus have reduced type 2 cytokine responses, suggesting that altered immune responses associated with infection may greatly impact drug studies.  

Normal Flora: The presence of normal flora is seldom confused with a drug-related effect, however, an increased burden may be present in immunocompromised animals. This should be documented, but care taken not to over interpret. 

Normal Flora and Immune Responses: The lower GI is the largest source of resident microflora. The upper GI is relatively sterile, while in the lower GI, a complex mix of microorganisms and gram-negative bacteria are required for normal immunological development.  These organisms are under strict regulatory control to avoid inflammation secondary to an immune response.  The role of normal flora in the development of the immune system has been demonstrated in germ-free animals. A role for commensal microflora and their immunoactivating components such as LPS, superantigens, and bacterial DNA has been suggested in the pathogenesis of complex diseases such as rheumatoid arthritis, IBD, allergies, colon cancer, periodontal disease, and atherosclerosis. Overgrowth of bacteria in the intestine can result in loss of mucosal integrity, suppression of immune function, and altered Kupffer cell responses.

Normal Flora and Drug Metabolism: Normal flora represent a unique metabolic system distinct from the metabolic activity of the host’s cells.  The metabolic potential of these flora changes with the host’s health.  Differences in drug metabolism may also be linked to variations in gut flora metabolism between individuals and within an individual over time.  Metabolic activity in the gut lumen can have an effect on the bioavailability and pharmacokinetics of drugs.  The large intestine, because of the large numbers of bacteria, has the greatest effect.  Diet changes also affect drug metabolism.  A high protein, low carb diet increases the metabolism of antipyrine and theophylline, whereas a low protein/high carb diet slows the metabolism of these drugs.  Food and food ingredients may alter drug metabolism with changes in the levels of cytochrome P450-dependent monooxygenases.  The gut microflora in macaques plays a significant role in the metabolism and pharmacokinetics of new drug candidates and contributes to interanimal variability.  

Selection and Screening of NHPs for Toxicology Studies: It is generally advisable to use macaques form one source to avoid regional differences in normal flora and pathogens.  Most important is to avoid mixing animals from different sources in one study.  For toxicology studies, animals should be selected on a SPF status, with screening for SRV, SIV, B virus, measles, HAV, and TB.  Fecal evaluation and bacterial cultures along with prophylactic antibiotics and antihelminthic treatment are useful in cases of sporadic diarrhea. For animals on studies with immunomodulatory drugs, certain opportunistic infections, like Plasmodium, can be diagnosed and treated before study initiation, while others that have a high prevalence but are not treatable can be documented so that animals can be distributed evenly among study groups.
 
QUESTIONS:
1. 	The most common primates used by pharmaceutical companies are?
2. 	Name the three retroviruses NHPs are typically screened for prior to drug studies.
3. 	What is typically the source of measles infection in NHPs? 
4. 	What is the pathogen associated with “bloody nose syndrome” in macaques?
5. 	What surgical procedure enhances susceptibility and infection of NHPs to malaria?

ANSWERS:
1. 	Cynomolgus and rhesus macaques
2. 	SRV, SIV, STLV
3. 	Infected human handlers
4. 	Moraxella catarrhalis
5. 	Splenectomy

Wachtman and Mansfield.  Opportunistic Infections in Immunologically Compromised Nonhuman Primates, pp. 191-208

SUMMARY: There are several endogenous diseases of primates that result in blunted immune response and allow for development of opportunistic infections.  This article discusses SRV, measles and SIV as causes of immunosuppression then reviews the major opportunistic infections.  

Causes of immunosuppression; Simian Type D Retrovirus or SRV is the most important immunosuppressive virus both in terms of risk to the colony and as a confounder to research.  It is transmitted horizontally, vertically and sexually by symptomatic and asymptomatic animals.  Clinical signs include diarrhea, weight loss, lymphadenopathy, splenomegaly and retroperitoneal fibromatosis.  SRV has wide cellular tropism and causes profound cellular depletion by unclear mechanisms.  SRV associated opportunistic infections (OI) include bacterial septicemia, cytomegalovirus, candidiasis, cryptosporidiosis and noma.  Diagnosis requires a combination of serology and virus isolation or molecular detection.

Measles virus (genus Morbillivirus, family Paramyxoviridae) spreads rapidly through both OWP and NWP colonies via the aerosol route.  Disease in OWPs is generally mild with fever, upper respiratory signs and maculopapular exanthema.  The disease in NWP can be severe with gastrointestinal signs and high morbidity and mortality.  Immunosuppression is associated with panleukopenia and profound lymphoid depletion in the thymus, LNs and spleen.  Immune deficits may persist for up to 6 months after infection.  OIs associated with measles include disseminated CMV, adenoviral and bacterial pneumonia and candidiasis.  Measles is acquired from contagious human handlers.  

Simian Immunodeficiency Virus (SIV) is a lentivirus that naturally infects African macaque species.  It is generally asymptomatic in African species but experimental infection of Asian macaque species results in an immunodeficiency syndrome similar to AIDS.  The similarities between SIV and HIV infection including tropism for CD4+ T lymphocytes and monocytes using CD4 and CCR5 chemokine receptors for viral entry, make SIV the best animal model of HIV infection.  

Viral Opportunistic Infections
1.	Cytomegalovirus
a.	OWP and NWP
b.	Transmission: oral secretions, breast milk, and urine
c.	Clinical Signs: diarrhea, respiratory compromise, neurologic sequelae
d.	TX: ganciclovir
e.	Histopath: nuclear and cytoplasmic enlargement with intranuclear (“owl’s eyes”) and intracytoplasmic inclusions
1.	Rhadinovirus (related to Kaposi’s sarcoma herpesvirus)
f.	Asian and African macaques, baboons, African greens, chimps and gorillas
g.	Transmission: oral secretions
h.	Clinical signs: fever, lymphoproliferation resembling multicentric Casteman’s disease,  retroperitoneal fibromatosis
i.	Histopath: hyalinized follicles surrounded by layers of loosely concentric lymphocytes
2.	Simian lymphocryptovirus (related to Epstein-Barr virus)
a.	Rhesus and cynomolgus monkeys
b.	Clinical signs: LCV-associated malignant lymphomas
c.	Oral hairy leukoplakia:  hyerpkeratosis, parakeratosis, enlarged acanthocytes and brick shaped inclusion bodies may be present
3.	Cercopithecine herpesvirus 1 (B virus)
a.	Asian macaque species
b.	Clinical signs: oral, genital ulcerations
c.	Dx: serology and virus isolation but have limitations – virus has latent form
d.	High zoonotic potential with high morbidity/mortality in humans
4.	Adenoviridae
a.	Respiratory and GI disease
b.	SIV – chronic active pancreatitis, necrohemorrhagic tubulointerstitial nephritis
5.	Papovaviridae = SV40
a.	Latent infection in Asian macaques first found in kidney cell lines used in polio vaccine production
b.	Clinical signs after SIV infection or immunosuppressives for organ transplant and include pulmonary, renal or CNS (similar to progressive multifocal leukoencephalopathy)
c.	Dx: serology or PCR of blood or urine (shed in urine)
6.	Parvoviridae= Simian parvovirus
a.	Tropism for erythroid lineage
b.	Clinical signs: profound anemia, diarrhea, weight loss, dehydration
c.	Dx: bone marrow – marked dyserythropoiesis, large intranuclear inclusions may be present

Bacterial Opportunistic Infections
1.	Mycobacterium avium complex
a.	Composed of M. avium and M. intracellulare
b.	Most common disseminated bacterial infection in both human and simian AIDS
c.	Source appears to be potable water and rarely occurs outside of HIV/SIV – see when CD4+ T cells fall below 200/µL
d.	Clinical signs: diarrhea, weight loss, peripheral lymphadenopathy, hepatosplenomegaly
e.	Dx: isolation of organism or PCR
2.	Rhodococcus equi
f.	Environmental contaminant
g.	Anorexia, weight loss and diarrhea, pyogranulomatous inflammation in lung, large intestine and draining lymph nodes
3.	Enteropathic E. coli (EPEC)
h.	Neonatal macaques – acute nonhemmorhagic diarrhea
i.	NWPs – acute hemorrhagic diarrhea
j.	Dx: colon biopsy, PCR of intimin (eaeA) gene
4.	Noma – polymicrobial infection (anaerobes and spirochetes)
k.	Rapidly progressive necrotizing stomatitis and osteomyolitis
l.	See in macaques infected with SIV and SRV
5.	Acute necrotizing gingivitis
m.	Borellia vincettii and Fusobacterium spp.
n.	Swollen bleeding gums covered by a characteristic pseudomembrane
o.	Clinical signs: malaise, fever and leukocytosis

Fungal Opportunistic Infections
1.	Pneumocystis carinii
a.	Clinical signs: pneumonitis, dyspnea, tachypnea, cough is uncommon
b.	Dx: PCR and cytology of cytospin prep from BAL
2.	Candida albicans
c.	Clinical signs: thrush
d.	Dx: Identify organisms with GMS or PAS stains, grows on Sabouraud and blood agar

Parasitic Opportunistic Infections
1.	Cryptosporidium parvum
a.	Severe symptoms with dissemination to liver, pancreas or respiratory tract
b.	Dx: fecal exam, colon biopsy, fecal antigen capture
c.	Chlorine and quats are not effective at eliminating from the environment -> chlorine dioxide, hydrogen peroxide, ethylene oxide and steam are effective
2.	Enterocytozoon bieneusi
d.	Invades hepatobiliary system causing jaundice and hepatomegaly
e.	Dx: PCR, indirect immunofluoresence, float with modified chromatrobe stain
f.	Can identify organisms via US-guided cholecystocentesis
3.	Acanthamoeba
g.	Infects both humans and animals as OI
h.	Healthy individual -> primary amoebic meningoencephalitis (PAM)
i.	Immunocompromised -> granulomatous amoebic encephalitis (GAE)
j.	Dx: post-mortem -> PAS or Geimsa confirms presence of organisms
4.	Toxoplasma gondii
k.	Callitrichids and owl monkeys are particularly sensitive
l.	Clinical signs are non-specific -> sudden death, neurologic disease, diarrhea, fever, cough, abortion
m.	Tx: clindamycin and TMS
n.	Exclude feral rodents from facility; cockroaches can also carry infectious oocysts
5.	Plasmodium
o.	Infection is nearly universal except in rhesus, callitrichids and Aotus
p.	Clinical signs: anorexia, cyclic fever, weight loss, anemia, leucopenia, thrombocytopenia
q.	Dx: organism present in blood smear, ELISA, fluorescent antibody test

QUESTIONS:
1.	A. What is the rational for development of viral SPF NHP colonies? B. What are the conventional SPF agents? C. What other viruses are eliminated in expanded SPF colonies?
2.	Which of the OIs are considered zoonotic agents?
3.	What are the proposed mechanisms of immunosuppression in SIV infection in macaques?

ANSWERS: 
1.	A. Viral OIs are significant confounders to research involving immunosuppression as many are endemic in monkey colonies.  Spread of viral diseases is difficult to prevent with changes in husbandry practices so active programs to eliminate certain viruses from colonies must be established.  B. Cercopithecine herpesvirus 1 (B virus), simian T lymphotropic virus (STLV), SIV and SRV. C. RRV (rhesus rhadinovirus), LCV, CMV, simian foamy virus, SV40.
2.	Possible:  RRV, adenovirus, simian parvovirus.  Zoonotic: B virus, SV40, mycobacterium avium complex, Candida, Cryptosporidium, Enterocytozoon, Acanthamoeba, plasmodium.
3.	Decreased numbers of circulating CD4+ T cells are the hallmark of SIV infection.  There are several mechanisms that reduce CD4+ T cells including virally mediated destruction of infected cells, destruction by virus specific cytotoxic T lymphocytes or natural killer cells, antibody-dependent cell-mediated cytotoxicity, activation-induced cell death and destruction of lymph node and splenic architecture leading to reduced regenerative power.

Haustein et al.  Nonhuman Primate Infections after Organ Transplantation, pp. 209-219

Domain 1: Management of Spontaneous and Experimentally Conditions T3. Diagnose disease or condition as appropriate T4. Treat disease or condition as appropriate

Species: Primary- macaque

SUMMARY: Nonhuman primates, especially macaques, have been used as models for solid organ transplantation.  This is mainly because their immune system is similar to human primates.  Post-operative management is complex, and involves the use of many immunosuppressive agents with concomitant risks of infection.  Diagnosis of infections is also complicated by the fact that many NHP hide signs of illness.  The authors' survey revealed that the incidence of infection in NHP transplant models is 14%.  It was found that many of these infections resulted from reactivation of viruses endemic to the species.  These viruses included cytomegalovirus, polyomavirus and Epstein-Barr virus.  Infections were sorted into 3 time frames:  early, intermediate and late infections.  Early infections included wound infections.  Intermediate infections were from 1-6 months post-transplant, and included latent infections with viruses and also mycotic infections.  Late infections resulted from community-acquired viruses and were mainly uncomplicated. 

QUESTIONS:
1.	T/F.  Samples from post-transplant NHPs with suspected infections should be treated with caution, due to risk of contamination of research samples and risks for personnel. 
2.  	T/F.  Treatment options for NHP with infections post- transplant must be balanced on one side with ethical treatment of the animal and value of research data from each animal on the other.
3.	T/F.  Drugs used to treat infections in NHP transplant recipients must be carefully checked for interactions with drugs that were used to induce immunosuppression in that animal.

ANSWERS:
1. 	T
2. 	T
3. 	T

Gardner and Luciw.  Macaque Models of Human Infectious Disease, pp. 220-255

Domain: Research
Task: Advise and consult with investigators on matters related to their research
Knowledge: Animal models (spontaneous and induced)

Species: Primary – Macaques

SUMMARY: Macaques have served as models for more than 70 human infectious diseases. With the exceptions of Chagas disease and bartonellosis, all the infectious diseases in this review are of Old World origin. 

Macaques are the major NHP resource for biomedical research. The NIH-supported National Primate Research Centers supply and house about 40,000 NHPs to investigate human disease.

Fun fact: The macaque genome sequence (Rhesus Macaque Genome Sequencing and Analysis Consortium 2007) is now available.

AIDS/SIV (Retroviridae): Simian immunodeficiency virus is a lentivirus, closely related to human immunodeficiency virus.

Fun fact: SIV does not occur in Asian macaques in the wild, but was acquired in captive macaques by accidental or purposeful cross-species infection from sooty mangabeys or  African green monkeys.

In natural African hosts, SIV strains are asymptomatic and transmitted mainly through wounds or bits acquired in fights or through sexual contact. 

The major difference in the host-virus relationship between African monkey and Asian macaques is the presence in the latter of a much stronger anti-SIV immune response associated with T and B cell activation and a loss of CD4+ T lymphocytes.  In the different species of macaques tested, SIV (mainly of sooty mangabey origin) causes an AIDS-like disease with remarkable similarities to HIV-1/AIDS. However, the SIV-induced macaque disease usually has a much shorter incubation period (2 months to 3 years) than does HIV-induced AIDS in humans.

Fun fact: The HIV-1 pandemic almost certainly arose by cross-species spread of SIV from chimpanzees via the bushmeat trade.

The value of the macaque model for AIDS research is the lack of persistent infection and development of signs of immunodeficiency in macaques experimentally infected with HIV-1. SIV is the experimental system of choice for comparative research on HIV/AIDS. The molecular make-up pathogenesis, pathology of SIV and HIV are similar in the respective susceptible host.

The infection of macaque species with SIV of initial sooty mangabey origin remains the animal model of choice for AIDS research.

Although HIV-1 does not productively infect macaques, chimeric viruses, designated simian/human immunodeficiency virus (SHIV), bearing HIV-1 envelope or reverse transcriptase genes, do so and can induce SAIDS. It is possible to test therapies and vaccines targeted to HIV gene products in the macaque. An important anti-HIV drug, tenofovir, was developed in the SAIDS model.

Influenza/Influenza virus (Orthomyxoviridae): Macaques are susceptible to influenza A virus, but the principal animal models for extensive immunization trials and strain typing remain ferrets and mice.

Macaques are used to compare the pathogenesis of highly virulent influenza strains, such as the reconstructed 1918 pandemic strain and the pathogenic bird flu strain (H5N1), with a nonvirulent conventional strain (H1N1).

Hepatitis A/Hepatitis B Virus (Hepadnaviridae): Natural infection with HAV, associated with mild clinical disease, has been detected in newly caught macaques.
Natural infection with HBV-like virus, associated with mild hepatitis, was observed in recently imported cynos from Indonesia. Due to highly effective HAV and HBV vaccines, there is no need for an animal model for these viruses.

Hepatitis C virus (Flaviviridae): Cynos, rhesus, and Japanese macaques are resistant to experimental HCV infection: therefore, the chimpanzee, which is susceptible, remains the animal model of choice for HCV vaccine model for HCV immunogenicity studies.

Hepatitis E virus (Hepeviridae): Emerging human pathogen spread by the fecal-oral route. Macaques are susceptible to natural infection, may transmit the virus to humans, and are used in vaccine studies.

Hepatitis G virus (Flaviviridae): Transmitted via blood transfusion. Small number of rhesus are infected. Since no evidence of harm to humans, no need to develop anti-HGV vaccines, tests, or drugs.

Tuberculosis (Mycobacterium tuberculosis): Macaques are highly susceptible to human M tb, as well as bovine and avian Mycobacteria, and manifest the complete spectrum of clinical and pathologic signs of human TB. Natural outbreaks of human, bovine, or avian TB occurred repeatedly over the years in captive macaques. Macaques were used to demonstrate the efficacy of the bacillus Calmette-Guerin (BCG) vaccine, an attenuated strain of M. bovis. The macaque model is helping to better define immunodominant antigens, conserved in different M tb strains. The macaque model can also be useful for the study of coinfection with SIV. The macaque model can lead to a better understanding of M tb pathogenesis.

Gastritis/Gastric Cancer (Helicobacter pylori): The macaque is a great animal for investigating the pathogenesis of H. pylori-associated gastritis and gastric cancer. They develop erosions indistinguishable from those that occur in humans.

Fun fact: About half the world’s population is infected with H. pylori.

Poliomyelitis/Poliovirus (Picornaviridae): The Americas were declared polio-free in 1994, but polio is still a concern in Africa and Asia. Use of the macaque is therefore still necessary for monitoring the potential neurovirulence of the live attenuated Sabin polio vaccine.

Fun fact: Humans are the only known natural host for poliovirus.

Fun fact: Simian vacuolating virus 40 (SV40), a polyomavirus, was discovered in 1959 as a contaminant of inactivated poliovirus vaccine prepared in macaque cell cultures. However, there is no conclusive evidence to implicate SV40 virus of polio vaccine origin in any human disease.

Measles (Paramyxoviridae): Measles is the leading cause of vaccine-preventable childhood mortality world-wide, responsible for about 345,000 deaths in 2005. Experimental inoculation of macaques with wild measles virus strains produces a disease that closely mimics human measles in its clinical and pathologic manifestations. These monkeys are used to test new vaccine strategies.

Chickenpox/Varicella zoster (VZV)/Simian Varicella virus (SVV) (Herpesviridae): Simian varicella virus (SVV) is partially genetically similar to human varicella (chicken pox) and herpes zoster (shingles) virus (VZV). SVV causes a highly contagious disease, often fatal, in Old World monkeys. There is no evidence that SVV infects humans. Like VZV, SVV establishes lifelong latent infection in neural ganglia, where it may be reactivated (e.g., after gamma irradiation).   Macaques offer an opportunity to evaluate new VZV vaccines, with improved efficacy and safety over present ones in use for humans.

Respiratory Syncytial Virus (RSV) (Paramyxoviridae): RSV is a major cause of severe respiratory disease in infant and elderly humans. The macaque model is now proving helpful for assessing the safety of novel RSV vaccines designed to prevent the post-vaccine enhanced disease that occurs after the individual had a prior RSV infection.

Metapneumovirus (Paramyxoviridae): This newly discovered human paramyxovirus is a causative agent of acute lower respiratory tract infection in very young children, the elderly, and immunocompromised patients. The virus is readily cultured in rhesus monkey kidney cells.

Cytomegalovirus (CMV) (Herpesviridae): Congenital CMV infection in humans is the leading infectious cause of birth defects in newborns. As in humans, macaque CMV is generally present after puberty as an asymptomatic lifelong infection. Reactivation of CMV, with associated inflammation, occurs in immunosuppressed humans and macaques, most dramatically evident in humans and simian AIDS. Macaques are used in vaccine development.

Herpes B Virus (Herpesviridae): B virus is an alphaherpesvirus that is now completely sequenced and is the macaque counterpart of herpes simplex virus (HSV) in humans. Coinfection of macaques with SIV seldom causes activation of latent B virus despite the frequent activation of other latent herpesviruses (e.g., CMV and Epstein-Barr virus). It is a zoonoses with a high incidence of encephalitis and death. The immunogenicity of a DNA vaccine against B virus has been tested in mice and uninfected rhesus macaques. Future vaccine combinations will include live vectors, such as vaccinia virus, aimed at eliciting cell-mediated immunity, which is important for protecting against B virus.

(Remember that the real name is Cercopithecine herpesvirus 1)

Human Herpesvirus 6 (HHV-6) (Herpesviridae): A macaque model may help in investigating whether HHV-6 might accelerate progression to AIDS in HIV-infected people.

Yellow Fever (Flaviviridae): Yellow fever virus was the first described human pathogen transmitted by an insect (Aedes aegypti mosquito). The natural source is monkeys. Vaccine testing relies on rhesus monkeys for proof of efficacy and safety testing.

Fun fact: In 1927 researchers gave macaques yellow fever from filtered human blood to successfully apply Koch’s postulates to a virus infection for the first time in virology. 

Fun fact: The first yellow fever vaccine was made in 1939 from the brains of experimentally infected macaques.

Dengue (Flaviviridae): Epidemic dengue is second only to malaria as the most important mosquito-borne (Aedes aegypti) disease affecting humans. Rhesus are used for vaccine development and pathogenesis, since they are quite susceptible to experimental infection. Asymptomatic infections in SE Asian macaques are documented.

(Clinical features of dengue include the sudden onset of fever, severe headache, myalgias and arthralgias, leukopenia, thrombocytopenia and hemorrhagic manifestations. Occasionally produces shock and hemorrhage, leading to death.) 

Chikungunya Virus (Togaviridae): Macaques are susceptible to experimental infection of this virus that is spread by mosquitoes (A. aegypti and A. albopictus) and has rapidly reemerged in Africa, India, Malaysia, and Italy. Monkeys used to study disease transmission and feasibility of vaccine creation.

Fun fact: The name is derived from the Makonde (Tanganyika) word meaning “that which bends up” in reference to the stooped posture that develops as a result of the disease’s arthritic symptoms.

Japanese Encephalitis (Flaviviridae): Japanese encephalitis virus (JEV) is one of the most important causes of viral encephalitis worldwide and is spreading throughout much of Asia. It is transmitted by Culex mosquitoes between wild and domestic birds and pigs. Macaques are used to improve upon existing vaccines

Rift Valley Fever (Bunyaviridae): This mosquito-borne tropical disease virus causes major morbidity and mortality in livestock and humans in sub-Saharan Africa. Rhesus are quite susceptible to experimental infection and 20% develop hemorrhagic disease. They are used to show the beneficial effects of antiviral drugs and alpha interferon and are suitable for developing new methods of diagnosis and therapy for the DIC that occurs in the severe disease.

Arenaviruses/Lassa Fever and Lymphocytic Choriomeningitis (LCM) Virus (Arenaviridae): Infection with LCM and Lassa virus has been modeled in macaques. Research has established the LCMV infection of macaques as a surrogate for Lassa virus infection of humans. Rhesus also used in Lassa virus vaccine trials.

Fun fact: LCM originated in the Old World, but is now worldwide in distribution. Lassa virus is localized to West Africa. 

Leprosy (Mycobacterium leprae): Experimental infection of rhesus with M. leprae caused clinical leprosy in about 50% of them. Naturally occurring leprosy has been documented in chimpanzees, sooty mangabeys, and wild-caught cynos.

Buruli Ulcer (Mycobacterium ulcerans): A major health problem causing disfiguring skin ulcers. Transmission may occur by biting water bugs and mosquitoes. Occurs in Australia, but primarily Africa. Cynos may be a valuable model for vaccine development.

Typhus (Rickettsiae): Louse-borne  rickettsiae cause epidemic typhus and scrub typhus (or fleas or chiggers). Cynos are susceptible to experimental infection and are the model in improvements to existing vaccines.

(This disease has affected the course of human history because of epidemics in armies and other crowded populations with poor hygiene. Symptoms include severe headache, a sustained high fever, cough, rash, severe muscle pain, chills, falling blood pressure, stupor, sensitivity to light, and delirium. A rash begins on the chest about five days after the fever appears, and spreads to the trunk and extremities but does not reach the face, palms and soles. A symptom common to all forms of typhus is a fever which may reach 39°C (102°F).)

Malaria (Plasmodia): The most important mosquito-borne disease affecting humans. Worldwide incidence is over 300 million cases with 1.3 million deaths annually. Of the four species of parasites infect humans, the most common are P. falciparum (avian origin) and P. vivax (macaque origin). Macaques are susceptible to  experimental infection with those, as well as 10 related plasmodium species. Antimalarial drugs successfully eliminate the parasitemia, but malarial parasites have developed resistance against some of these drugs. Macaques are used in vaccine development. Most research has focused on blocking the initial infection, using irradiated killed sporozoites, CS surface proteins recombined with hepatitis B virus surface antigen, adenovirus recombinants, or naked DNA in various combinations.

Schistosomiasis (Schistosomia mansoni): Schistosomia species (parasitic trematodes) infect about 250 million people, resulting in low mortality, but debilitating conditions. S. mansoni primarily affects the liver. Macaques are susceptible to experimental infection with S. mansoni. Rhesus are useful for understanding the immunology of schistosomiasis because they develop a solid immunity to reinfection.

Chagas Disease (Trypanosoma cruzi): Chagas disease occurs primarily in rural areas of South America and is absent in the Old World. Its pathogenic agent is a flagellate protozoan names T. cruzi, which is related to the agent of African sleeping sickness. The protozoa are transmitted to humans by the bites of triatomids (“kissing bugs”). A large natural reservoir for the organisms includes other infected humans, domestic animals, wild rodents, and monkeys. Both acute and chronic Chagas disease models have been established in rhesus.

Fun fact: Charles Darwin may have acquired this disease (i.e., cardiomyopathy) from the bite of what he called “a great black bug of the Pampas.”

African Sleeping Sickness (Trypanosoma brucei and T.b. rhodesiense): Rhesus macaques infected with T.b. rhodesiense develop a glomerulonephritis that is associated with activation of the alternate complement pathway. Stump-tailed macaques show neurologic signs. No vaccine. Diamidine is effective in eliminating parasites from the blood.

Acariasis: Stump-tailed macaques experimentally infected with the nematode Ascaris suum have served as a model for allergic bronchitis.

Lymphatic Filariasis: Rhesus serve as a model of filariasis, which causes massive lymphedema or elephantiasis.

Papillomavirus (Papillomaviridae): Human papillomaviruses (HPV) are the cause of cervical cancer in humans. About 50% of rhesus from several different primate facilities are infected with indigenous papillomaviruses. Macaques are natural hosts of genital papillomaviruses. Macaque cervical intraepithelial neoplasia resembles that of humans. The macaque papillomavirus model should prove valuable for further research into the pathogenesis, therapy, and prevention of cervical cancer.

Syphilis (Treponema pallidum): Macaques can clear an intrathecal inoculation of T. pallidum from the CNS, as do humans with early syphilis.

Chlamydia (Chlamydia trachomatis): The most common sexually transmitted disease in the US, with 1 million cases reported to the CDC. Can pass from an infected mother to her baby in the birth canal and causes trachoma, a disease characterized by conjunctivitis leading to blindness. Most men and women as asymptomatic. Macaques are useful models for each aspect of C. trachomatis infection.

West Nile Virus (WNV) (Flaviviridae): Macaques used in vaccine development. The vaccine is licensed for use in horses, but not yet in humans.

Hantaviruses (Bunyaviridae): Hantaviruses, maintained in rodent reservoirs, cause two severe human diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). Macaques used in vaccine development for both syndromes.

Severe Acute Respiratory Syndrome/SARS Coronavirus (Coronaviridae): Initial transmission of SARS-coronavirus to cynos helped to prove this virus is the primary cause of SARS. Vaccines show promising results in macaques.

Ehrlichiosis: Rhesus develop a classical granulocytic ehrlichiosis after IV inoculation with Ehrlichiae-infected blood and could be important for further study of diagnosis, management, and intervention of CGE.

Lyme Disease (Borrelia burgdorferi): Rhesus are used in pathogenesis studies.

Bartonellosis: A rhesus model was recently developed for trench fever (Bartonella quitana).

Smallpox/Variola Virus (Poxviridae): Smallpox virus, that may someday be stolen from frozen stocks in the US or Russia, poses a significant bioterrorism threat. Cynos are used to urgently develop vaccines and improved drugs.

Monkeypox (Poxviridae): Monkeypox virus causes a natural and experimental disease in cynos similar to human smallpox, and so this animal model can help to increase the effectiveness of antiviral drugs (like cidofovir) and candidate novel smallpox vaccines. Vaccinia virus immunization affords long-lasting protection of macaques against monkeypox virus challenge, except those with SAIDS.

Rabies (Rhabdoviridae): Because of the expense of the current vaccines, derived from cell cultures and problems with stability, efforts are underway to develop a subunit or DNA vaccine. Cynos are used.

Marburg and Ebola Viruses (Filoviridae): Marburg and Ebola Viruses cause epidemics of hemorrhagic fever and are among the most virulent viruses that infect humans. Wild NHPs and humans are accidental hosts. Ebola virus-infected monkeys have been studied to gain insight into the pathogenic mechanisms that lead to the profound lymphopenia, disseminated intravascular coagulation, and hemorrhagic and septic shock that are characteristic of filovirus infection of humans.  The coagulation abnormalities are most likely triggered by immune-mediated mechanisms rather than by direct damage to endothelial cells. Lymphopenia is attributed to infection of dendritic cells, blocking their maturation and thus inhibiting activation of lymphocytes and triggering bystander apoptosis. Release of tissue factor from monocyte/macrophage is a key event in triggering disseminated intravascular coagulation (DIC) in Ebola virus-infected macaques. Macaques are used in vaccine and treatment studies.

Anthrax (Bacillus anthracis): Aerosolized B. anthracis spores are considered the foremost infectious biological threat in the US. It is important to prepare and stockpile an anthrax vaccine. Macaques are a great model for testing vaccines and drugs because they are quite susceptible to experimental B. anthracis inhalation-induced disease. Macaques are also used for testing antibodies against inhalation anthrax given either alone or together with the vaccine.

Tularemia (Francisella tularensis): Tularemia is serious and often fatal. Outbreaks of naturally acquired tularemia have also been described in NHPs, including macaques that can develop the full-blown pneumonic form.

Plague (Yersinia pestis): The human-to-human spread presents a bioterrorism threat and has prompted efforts to develop a plague vaccine using monkeys.

Brucellosis (Brucella): This coccobacillus is a highly infectious biological warfare threat. Vaccinated macaques were solidly protected from aerosol challenge.

Simian T Lymphotropic Virus (STLV) (Retroviridae): The natural history and biology of STLV are very similar to those of human TLV. However, STLV-infected macaques do not serve as a practical model for HTLV infection because the latent period is long (40-50 years) and the tumor incidence is below 1%. Unlike HTLV, STLV has not been linked to lymphomas, immunosuppression, or neurological diseases in macaques. A more suitable animal model for HTLV-associated lymphomagenesis is a baboon infected with STLV and Epstein-Barr Virus.

Simian Type D Retrovirus (SRV) (Retroviridae): Simian Type D retrovirus is an indigenous retrovirus of macaques that can cause outbreaks of an immunosuppressive and fatal wasting disease in cohoused animals. 

Fun fact: The associated disease was at first called simian AIDS (SAIDS), but this term is now confined to the SIV-induced disease in macaques and an occasional African NHP species.

SRV differs from SIV both in its pathogenesis and in being more distantly related to HIV (SRV is not a lentivirus.) Unlike SIV and HIV, SRV causes a generalized infection of both T and B cells, is genetically stable, and can be controlled by neutralizing antibodies, whether induced naturally or by vaccines. SRV-infected macaques are the major source of a Kaposi’s sarcoma (KS)-like lesion called retroperitoneal fibromatosis (RF). In contrast to SIV, SRV infection is not associated with B cell lymphoma or activation of latent EBV-like virus. Simian Type D Retrovirus is an important model for retrovirus-induced immunosuppression. Also, SRV must be eliminated in NHPs used for research, particularly in studies of SIV-induced SAIDS.

Epstein-Barr Virus (EBV)/Lymphocryptovirus (Herpesviridae): Macaques harbor a homologue of human EBV whose biology in macaques appears identical to that of EBV in humans. Like human acute EBV, the macaque virus generally causes a lifelong, asymptomatic, latent infection. Key aspects of human acute EBV infection are reproducible in the macaque model, including oral transmission, atypical lymphocytosis and lymphadenopathy, and activation of B cells, all features of infectious mononucleosis. Activated EBV in macaques also causes oral lesions (called hairy cell leukoplakia) that manifest in human AIDS.

Kaposi’s Sarcoma-Associated Herpesvirus (Herpesviridae): Two distinct lineages of rhadinovirus-RV-1, or retroperitoneal fibrosis (RF)-associated herpesvirus (RFHV), and RV-2, or rhesus rhadinovirus (RRV), a more distantly related virus – are related to Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV, human herpesvirus 8), the causative virus of KS in humans. Experimental infection of macaques with RRV alone or together with SIV has induced generalized lymphadenopathy in a few animals, but as yet no RF lesions or lymphomas. Because RRV replicates at a very low levels and induces no specific pathologic lesions, it is unlikely to provide a useful animal model for disease.

Rotaviruses (Reoviridae): Group A rotaviruses are ubiquitous and infect most humans by the 3rd year of life. These viruses are the single most important cause of severe diarrhea in infants and young children worldwide. NHPs, including macaques, are naturally infected with simian rotavirus strains, but in the natural simian host diarrhea is usually not manifest. Newborn macaques also are susceptible to experimental infection and diarrheal disease from human rotavirus isolates.

Norwalk Virus (Noroviridae): About half of the US outbreaks of acute infectious nonbacterial gastroenteritis are due to the Norwalk virus. Antibodies against the virus are not protective. Macaques are susceptible to experimental infection, but do not exhibit clinical gastroenteritis.

Tick-Borne Encephalitis (TBE) (Flaviviridae): Tick-borne encephalitis is a nontropical zoonotic disease of increasing incidence in Asia and Europe. Rodents are the maintenance host, and TBE has been transmitted to dogs and horses as well as humans. Rhesus were used in vaccine development.

Simian Hemorrhagic Fever Virus (Arteriviridae): This macaque disease could be a model for studying the pathogenesis of viral hemorrhagic fevers in humans.

Simian Parvovirus/Erythrovirus (Parvoviridae): Simian parvovirus in macaques is used as a model for human B-19 parvovirus, which causes anemia, abortion, and arthritis in humans. SPV must be considered a potential zoonosis, since it has the ability to replicate in human bone marrow cells in vitro and causes viremia in infected macaques.

Polyomavirus (PV) (Polyomaviridae): SV40, the prototype polyomavirus (PV) indigenous in macaques, is analogous to the BK and JC PVs in humans. In humans with AIDS and macaques with SAIDS, activation of JC or SV40 virus respectively, causes demyelinating CNS disease, called progressive multifocal leukoencephalopathy. The SV40 natural strain is genetically different from the laboratory SV40. A PV recovered from immunosuppressed cynos (on a tissue transplant project), called CPV, caused interstitial nephritis in many of the grafted and native kidneys. CPV is an excellent model for the BK polyomavirus-induced interstitial nephritis and ureteral stenosis that occur in human renal transplant recipients.

Q fever (Coxiella burnetii): Cynos are used in vaccine development against aerosol infection.

Escherichia coli: Toxigenic strains of E. coli (e.g., 0157:H7) are causes of human morbidity and mortality. Macaques have served as models to investigate the pathogenesis and treatment of experimental E. coli infection for over 30 years.

Listeriosis (Listeria monocytogenes): Cynos are used as a model for infections.

Bacillary Dysentery (Shigellosis) (Shigella flexneri): Rhesus have served as models to investigate the pathogenesis of colonic mucosal invasion.

Streptococcal Pneumonia (Streptococcus pneumoniae): Attempts are underway to develop a rhesus model for pneumococcal vaccine assessment.

Streptococcal Pharyngitis (Streptococcus pyogenes): Cynos are used to study the pathogenesis of Group A streptococcal pharyngitis and the resultant rheumatic heart disease.

Toxoplasmosis (Toxoplasma gondii): Natural infection occurs in macaques living in human environments where domestic cats roam. Experimental infection was used to evaluate treatment in eliminating the parasite in both the amniotic fluid and neonate.

Periodontal Disease: The clinical, pathological, microbiological, and immunological characteristics of spontaneous and experimental periodontitis in macaques are similar to those of humans.

QUESTIONS:
1.	What is the SIV-induced macaque disease incubation time?
2.	How does this compare with HIV-induced human disease incubation time?
3.	Why is the chimpanzee the Hepatitis C model of choice and not macaques?
4.	T or F: The H. pylori gastric erosions are significantly different in humans and macaques.
5.	Why type of immunity is important for protection against Cercopithecine herpesvirus 1?
6.	What is the natural source of the Yellow Fever virus?
7.	What virus is a good model for Lassa virus infection of humans?
8.	T  or  F: The macaque papillomavirus model is an appropriate one for humans.
9.	For which hantavirus syndrome are macaques used for vaccine development, the hemorrhagic fever with renal syndrome or hantavirus pulmonary syndrome?
10.	Why might cynomologous macaques be needed for smallpox vaccine development and improved treatments?
11.	What is the most likely cause of coagulation abnormalities in Marburg and Ebola infection in humans and monkeys?
12.	Why is Simian T Lymphotropic Virus infection in macaques not a good model for Human T Lymphotropic Virus?
13.	How does Simian Type D Retrovirus differ from SIV and HIV?
14.	What is the term for oral lesions in macaques that are caused by the Epstein-Barr  Virus?
15.	Why is Simian Parvovirus considered to be a potential zoonosis?
16.	SV40 is analogous to what polyomaviruses in humans? 

ANSWERS:
1.	2 months to 3 years
2.	Much shorter
3.	Cynos, rhesus, and Japanese macaques are resistant to experimental HCV infection.
4.	False:   The lesions are indistinguishable.
5.	Cell-mediated immunity.
6.	Monkeys.
7.	Lymphocytic Choriomeningitis Virus in macaques.
8.	True
9.	Both
10.	Because of the risk of frozen stock theft by terrorists.
11.	Immune-mediated mechanisms.
12.	The latent period is of STLV long and tumor incidence is below 1%.
13.	SRV causes a generalized infection of both T and B cells, is genetically stable, and can be controlled by neutralizing antibodies, whether induced naturally or by vaccines.  
14.	Hairy cell leukoplakia
15.	The SPV can replicate in human bone marrow cells in vitro.
16.	B